Oral health is essential for social and emotional well-being, making dental hygiene a daily priority for many. The most common products – toothpastes and mouthwashes – are designed to combat bacteria, plaque and food residues, either mechanically or through rinsing. Among the beneficial ingredients in oral care are polyols like sorbitol, xylitol and erythritol, which offer sweetness, improved taste and valuable prophylactic properties against oral biofilm.
Introduction
A broad smile makes a good first impression in every face-to-face encounter and plays a vital role in communication. Not surprisingly, healthy teeth are perceived as an important positive visual trait. Several studies have linked oral health with social and emotional well-being. [1,2] However, in our fast-paced society, people often have little opportunity to brush their teeth frequently or evaluate the impact of the food they consume on their dental health. This increases the need for products and ingredients which support and prolong the effect of oral care products.
The most commonly used products are toothpastes and mouthwashes. Newer formats such as tooth tablets or powders follow the same principle, being designed to remove bacteria, plaque and food residues either mechanically or by rinsing. The substance commonly referred to as plaque or oral biofilm is actually an agglomeration of microorganisms, usually bacteria, embedded in and mechanically protected by a matrix of polymers. [3]
The formation of such a biofilm starts when bacteria gain initial adhesion on a pellicle. This pellicle serves as an anchor point on the surface of the tooth: the bacteria then start to grow and multiply, they communicate with each other, and they secrete glycoproteins, resulting in a biofilm. Biofilms have been reported to be associated with dental caries, periodontal disease and gingival conditions. [4] It is therefore a common aim of oral care products to reduce the level of biofilm in the oral cavity. The most obvious way to do this is by mechanical removal (brushing), but supporting ingredients that impact bacterial growth can also contribute to this objective.
Different bacterial species are present in a healthy oral microbiome. This is not dangerous per se, nor will it cause any pathological condition as long as the bacteria present are in homeostasis. However, if conditions in this environment change – for example, increased sugar availability after eating – certain species will respond to their new environment differently from others. Clinical studies have shown that individuals with dental caries also demonstrate increased proportions of acidogenic and aciduric bacteria (especially mutans streptococci and lactobacilli), which are capable of demineralising enamel. [5] These bacteria do so by metabolising dietary sugars to acid, which decreases the local pH, generating an acidic environment which is favourable for their growth but disadvantageous for other species.
Reducing sugar consumption and applying oral care products with ingredients which inhibit the growth of unwanted bacteria are therefore two of the main pillars in oral biofilm prophylaxis. One class of ingredients which have proven beneficial are polyols such as sorbitol, xylitol and erythritol. This study presents evidence of the positive impact of erythritol on oral biofilm in realistic scenarios.
General properties of erythritol
Erythritol (brand name ERYLITE®) is a white, crystalline and odourless product widely used in food, beverages and personal care products. It is a natural sweetener approximately 60% as sweet as sugar. It can be considered natural because it occurs in nature and the production process is based on a non-genetically modified yeast fermentation of plant-based raw materials.
Erythritol is a four-carbon sugar alcohol (polyol) with a very low molecular weight (122 g/mol) and is characterised by unique metabolic attributes: after consumption, the vast majority is absorbed in the small intestine, and is therefore excreted in the urine instead of being metabolised. Consequently, it is tolerated very well by the digestive system. Furthermore, it delivers no calories, is non-glycaemic and does not trigger an insulin response.
Erythritol as a sustainable ingredient
The main raw material for erythritol production at Jungbunzlauer (ERYLITE®) is glucose derived from corn. Upon request, the glucose can be supplied by farmers committed to sustainable agriculture, as verified by the SAI Platform’s Farm Sustainability Assessment (FSA).
Jungbunzlauer’s sustainability commitments include:
- Jungbunzlauer is committed to the Science Based Targets initiative (SBTi) and sets targets to reduce greenhouse gas emissions in line with climate science – for example, by further improving the company’s energy management system.
- Jungbunzlauer regularly calculates product carbon footprints (PCFs) in collaboration with a certified and independent external partner (myclimate).
- The production plant for ERYLITE® is ISO 50001 certified and has implemented an energy management system focused on continuous improvement.
Benefits of erythritol in oral care
When erythritol is added to oral care products, its natural sweetness helps improve consumer perception by improving the taste of the product. It also allows formulation of innovative products with special cooling effects – e.g. toothpaste with crystals. [6]
In general, polyols are known for their non-cariogenic properties, since they cannot be metabolised by certain unwanted bacteria in the oral cavity. This is due to their molecular structure and the absence of reducing carbonyl groups, unlike sugars such as glucose or fructose. Polyols also reduce the growth of some bacteria which are associated with pathogenic conditions. [7,8] This is reflected in a statement by the European Food Safety Authority (EFSA), which recognises that the use of polyols as a sugar substitute in food products helps to maintain tooth mineralisation. [9]
While xylitol is probably the most prominent polyol used for oral health, an increasing number of recent studies have highlighted the potential of erythritol to act in the same way. [6] In vitro, erythritol was shown to have an impact via several pathways – including bacterial gene expression [10], bacterial metabolism [11], and accumulation of extracellular matrices. [11,12] Because of its small molecular size, erythritol was shown to readily penetrate biofilm, and therefore also has the potential to support the effect of antimicrobial compounds such as chlorhexidine. [13]
In vivo, the application of erythritol resulted in a reduction of dental plaque, delayed the development of caries, and reduced the overall occurrence of caries in a long-term study, which was also confirmed in a follow-up study. [14–16] De Cock et al. have published a review providing a broad overview of existing literature on relevant in vitro and in vivo studies, as well as clinical trials with erythritol. [17]
Erythritol also has other applications worthy of note, one of which is in tooth air-polishing powders used by dentists for professional prophylaxis. Over the last decade, air polishing with low-abrasive erythritol-based powder has become increasingly popular. This is a rapid procedure which achieves maximum cleaning performance and is more convenient for patients than traditional alternatives. [18]
Another application of erythritol is in oral care products for animals. Like humans, pets also suffer from bad breath and periodontal diseases. Products to improve oral health in pets, such as dog chewing sticks containing erythritol, are becoming increasingly widely available. The literature shows that erythritol provides the same advantages for canines as it does for human oral health [19,20], and – in contrast to xylitol – erythritol is safe for use in animal care products, being non-toxic to dogs and other popular pets. [21,22]
To sum up, the literature shows that polyols in general, and erythritol in particular, have a positive impact on oral health and specifically on biofilm formation. However, the setups of the various studies differ widely, and the results are not readily comparable. Past studies have often involved exposure to polyols over extended periods of several hours, which does not adequately reflect use in oral care products. Furthermore, many tests have included elevated polyol concentrations, which are difficult to achieve in the oral cavity with oral care products due to factors such as space limitations or solubility limits in full formulations. Our study therefore aimed to better consider these aspects, further demonstrating the potential of erythritol in oral care products.
Study design to compare erythritol and xylitol on biofilm growth
Overall Objectives of the Study
The goal of the study was to compare the effects of erythritol and xylitol on biofilm growth of Streptococcus mutans in an in vitro model, using realistic exposure times and concentrations (Figure 1). Each polyol was subjected to two tests. The first test aimed to create a scenario mimicking tooth brushing, with a concentration of erythritol in toothpaste or tooth tablets of 10 wt%, a 1:2 dilution by saliva, and a typical tooth-brushing time of 3 minutes. The second scenario aimed to mimic mouthwashing, with a 10% concentration of polyol in the mouthwash product, no dilution, and a mouth-rinsing time of 1 minute. In both tests, sucrose was supplied after treatment with polyols to mimic consumption of detrimental cariogenic substances after brushing or mouthwashing.
The results were evaluated using the following methods:
- Plate counting of colony-forming units (CFUs) to determine bacterial viability
- Luminescence assay to measure adenosine triphosphate (ATP) as an indicator of bacterial metabolic activity
- Fluorescence microscopy to assess bacterial membrane integrity
Materials
All trials were performed by the Fraunhofer Institute for Microstructure of Materials and Systems (IMWS) (Halle an der Saale, Germany). The gram-positive bacterial strain Streptococcus mutans DSM‑20523 was chosen as the model organism and causative agent for the dental biofilm.
Polymethylmethacrylate (PMMA) discs with a slight abrasion on one side were used as a substrate for biofilm growth. Treatment solutions were freshly prepared in distilled water immediately prior to application. ERYLITE® was provided by Jungbunzlauer. Xylitol, Todd Hewitt broth, maximum recovery diluent (MRD), and Tween® 80 were obtained from Carl Roth (Karlsruhe, Germany). Sucrose and Fluoromount™ mounting medium were obtained from Sigma‑Aldrich (St. Louis, MO, USA).
Experimental Procedure
To prepare the model biofilm, S. mutans was cultivated for 24 h and the cell concentration was adjusted to an optical density (OD₆₀₀) of 0.1. The cell suspension was diluted 200‑fold using Todd Hewitt broth supplemented with 1% sucrose to ensure an adequate nutrient supply for biofilm growth, as suggested by Loimaranta et al. [23].
One millilitre of this inoculum was pipetted into each well of a 24‑well plate containing the PMMA discs and incubated for 16 h at 37 °C to allow the biofilm to settle and establish. The supernatant was then removed, and the discs were rinsed with MRD solution and transferred to a new well for treatment application.
Reflecting the different application scenarios described above, the following treatments were applied. Each test used a volume of 1 mL and was performed in triplicate:
- Negative control (distilled water)
- 5% erythritol for 3 min (tooth‑brushing scenario)
- 5% xylitol for 3 min (tooth‑brushing scenario)
- 10% erythritol for 1 min (mouthwash scenario)
- 10% xylitol for 1 min (mouthwash scenario)
After the relevant exposure times, the treatment solution was removed and replaced with 1 mL of Todd Hewitt broth containing 1% sucrose to mimic sugar consumption after brushing or mouthwashing and thereby renew the nutrient supply to the bacterial biofilm. After incubation at 37 °C for 3 h, the samples were rinsed again with MRD and transferred to a new well.
The biofilm was mechanically removed from the surface of the PMMA discs using a cotton swab, and the cells were resuspended in MRD solution containing 0.1% Tween® 80 by vortexing.
Quantitative evaluation by viable plate counting
Colony‑forming units (CFUs) were determined from the recovered cell suspension by performing a ten‑fold serial dilution. The diluted suspensions were plated on plate count agar and incubated at 37 °C for 72 h. Plates were prepared in triplicate.
Colonies were counted from digital photographs using the Cell F software package (Olympus, Germany). All plates containing between 10 and 400 colonies were included in the CFU calculation.
Qualitative evaluation by fluorescence microscopy
After rinsing the biofilm grown on the PMMA discs, cell staining was performed by applying Syto® 9 and propidium iodide dyes (LIVE/DEAD® BacLight™ Bacterial Viability Kit, Thermo Fisher Scientific) to the wells and incubating according to the manufacturer’s instructions.
These dyes selectively penetrate bacterial cells depending on membrane integrity. As a result, cells with damaged membranes appear red under a fluorescence microscope, whereas cells with intact membranes appear green.
The samples were mounted on microscope slides using Fluoromount™ mounting medium and analysed using a BZ‑X fluorescence microscope (Keyence, Germany) at 40× magnification.
Quantitative evaluation of metabolic activity
Biofilm samples were cultivated and treated with the test solutions as described above. After rinsing the PMMA discs, the reverse side of the discs was wiped with a paper towel, and the discs were transferred with the biofilm side facing down into a fresh well containing MRD and reagents from the BacTiter‑Glo™ Microbial Cell Viability Assay kit (Promega, Madison, WI, USA).
Samples were incubated and agitated for 12 min at room temperature. The BacTiter‑Glo™ reagents promote cell lysis to liberate the ATP present in the cells at a given instant and generate a luminescent signal that is proportional to the amount of ATP present. A calibration curve was prepared using an ATP standard.
After incubation, samples were transferred in duplicate to an opaque well plate, and luminescence was measured using a FLUOstar® Omega plate reader (software version 570R2, BMG Labtech, Ortenberg, Germany).
Statistical analysis
Statistical analyses were performed using one‑way analysis of variance (ANOVA), with post‑hoc Tukey’s tests and Levene’s tests to assess homogeneity of variance (Origin2020, OriginLab Corporation, Northampton, MA, USA). The level of significance (α) was set at 0.05.
Results and discussion
All experimental scenarios confirmed that erythritol and xylitol inhibit the growth of Streptococcus mutans.
Figure 2 shows the number of colony‑forming units (CFUs) in samples treated with polyols for each of the different application scenarios. As the gold standard of viable cell analysis, colony counting provided reliable insight into the ability of S. mutans to proliferate.
All polyol treatments resulted in a statistically significant reduction in CFUs. In the case of ERYLITE® (1 min exposure), CFU counts were reduced from 8.15 × 10⁸ in the control group to 4.62 × 10⁸, corresponding to a 53% reduction in cell viability. No statistically significant differences in growth inhibition were observed between the individual treatments.
[GRAPH 2]
As a complementary method, the luminescence assay – based on detection of ATP – fully backed up the results obtained by colony counting. Figure 3 shows how the different treatment scenarios reduced the ATP content from 97.3 pmol in the control to as low as 68.4 pmol (10% erythritol for 1 min). All polyol treatments resulted in a statistically significant reduction of the ATP content compared to the control, but there was no statistically significant difference between the treatments.
[GRAPH 3]
Qualitative analysis was undertaken by examining the samples using a fluorescence microscope after live/dead staining. As shown in figure 4, the proportion of red-stained cells – i.e. cells with compromised membranes – was larger in the samples treated with polyols than in the control. Although not significant, there was a slight trend indicating a dose-dependent effect.
[GRAPH 4]
The tested polyol concentrations of 5% and 10% were well within the range of concentrations previously investigated in the scientific literature and resulted in comparable reductions in cell viability. For example, Loimaranta et al. observed a reduction in Streptococcus mutans CFUs of approximately one order of magnitude (observed reduction: 90%) when the growth medium contained 5% erythritol or xylitol. [23] Kõljalg et al. reported growth inhibition of S. mutans in the range of 30–50% with erythritol or xylitol concentrations of 3.75–7%. [24] Staszczyk et al. measured growth inhibition in a similar range (below one order of magnitude) using polyol concentrations of 5%. [25] Finally, Ghezelbash et al. observed reductions of S. mutans viability of 68% and 71% when using 4% xylitol and 4% erythritol, respectively. [12]
Furthermore, it should be noted that strain‑specific susceptibility to polyol treatment has been described in the literature, indicating that the potential benefit of erythritol and xylitol application may depend on the patient’s pathogen profile. [7,24]
One notably different parameter in the present study is the short exposure time of the biofilm to polyol treatments. In the studies cited above, growth curves were recorded under continuous exposure to polyols for periods ranging from 10 h [23] to 72 h. [25] Typical oral hygiene routines, however, last between 30 s and 1 min in the case of mouthwashing, or between 1 and 3 min for tooth brushing. Consequently, experimental conditions in which biofilms are exposed to polyols for several hours do not adequately reflect real‑world use.
To the best of our knowledge, these results are the first to demonstrate that erythritol and xylitol provide significant benefits even after very short contact times of as little as 1 min.
A proposed mode of action by which erythritol and xylitol may impede biofilm formation is the reduction of exopolysaccharide production, resulting in less tightly adhering biofilms. [10–12,23] This may facilitate biofilm removal during rinsing and account for both the reduced cell counts and lower ATP levels observed in the present study. In contrast to harsh antimicrobial agents, polyols act more gently on the beneficial oral microflora, which plays an important role in both oral and general health.
Indeed, a long‑term study investigating the oral microbiome composition of children following regular polyol consumption demonstrated that erythritol induced a significant microbiome shift, resulting in a lower prevalence of caries‑associated mutans streptococci. [26] In addition, a recent study evaluating the biocompatibility of toothpaste formulations containing erythritol and xylitol confirmed their exceptional mildness toward oral mucosal cells; only a limited number of commercially available products met this criterion. [27]
Conclusion
The present study demonstrates the effectiveness of erythritol in an experimental setup designed to reflect realistic exposure times and concentrations. Growth inhibition of oral disease‑associated bacteria was confirmed using viable plate counting, fluorescence microscopy and luminescence‑based metabolic activity assays. The performance of erythritol was comparable to that of xylitol.
Erythritol is therefore suitable for use in a broad range of oral‑care product formats, including mouthwashes, toothpastes, gels and tablets. It represents a valuable natural ingredient that not only provides sweetness but also exerts a positive impact on oral biofilm formation.
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